Technical Abstract:
The 31-kDa peroxisomal membrane protein is a novel isoform of ascorbate peroxidase (pAPX) that likely functions in the regeneration of NAD+ and protection of the cell against toxic reactive oxygen species generated during postgerminative growth. In this study, the intracellular sorting of pAPX to the boundary membrane of glyoxysomes was examined in BY-2 cells. Both transiently-expressed, epitope-tagged pAPX and a chloramphenicol acetyltransferase (CAT) pAPX fusion protein (CAT-pAPX) were localized in vivo to glyoxysomes and to a subdomain of the endoplasmic reticulum (ER) termed herein as 'glyoxysomal ER' (gER). Results from mutagenesis experiments revealed the C-terminal portion of pAPX contains the topogenic information both necessary and sufficient for subcellular targeting. Application of brefeldin A (a fungal toxin that blocks protein export from the ER) interfered with the sorting of CAT-pAPX to glyoxysomes. In vitro experiments provided direct evidence that pAPX inserted specifically into ER membranes in a post-translational (SRP-independent) mechanism upon the presence ATP and molecular chaperones, and possibly a membrane-bound receptor. pAPX was not efficiently inserted into peroxisome membranes in vitro. Collectively, these results suggest that pAPX, a tail-anchored (Ncytosol-Cmatrix) integral membrane protein, is targeted to glyoxysomes via a pre-glyoxysomal compartment that is derived from a portion of the ER.